A stable kasugamycin technical, mother drug and composition thereof

Abstract

The present application provides a stable kasugamycin technical, a mother drug and a composition thereof. By controlling the mass ratio of kasugamycin or its salt to protein in the kasugamycin composition or controlling the protein content or the polysaccharide content in the kasugamycin composition, the kasugamycin composition has better stability in terms of odor, color appearance and / or hygroscopicity, and thus has significant beneficial effects in storage, freight and terminal applications. In addition, controlling key substances that affect stability guides production and manufacturing, which is beneficial to cost control.

Description

Technical Field

[0001] The invention relates to the field of pesticides, and in particular to a stable kasugamycin technical, a mother drug and a composition thereof. Background Art

[0002] Kasugamycin is highly effective, low-toxic and environmentally friendly, so it is widely used in the prevention and treatment of various plant diseases. Kasugamycin products will have a noticeable odor after being stored for a period of time, especially in liquid preparations. The instability of the product seriously affects the use of pesticides. In order to overcome the above problems, a common method is to add preservatives such as sodium benzoate to the product to inhibit the generation of odor, but it requires systematic research on the compatibility of preservatives and fungicides, which also increases the company's production costs and the difficulty of preparation production. There is also a risk of preservative residues on crops.

[0003] In addition, the applicant also found that kasugamycin products are prone to non-enzymatic color change during storage, which makes the appearance of kasugamycin products appear light yellow or brown. The visual sense of the products after color change is poor, and farmers think that the products are spoiled and dare not use them. The non-enzymatic color change reaction process is extremely complicated, and there is still no clear mechanism explanation. The control methods of non-enzymatic color change reactions in the actual production of pesticides are also limited. Some people add browning inhibitors to the products to prevent the discoloration of kasugamycin products, but the additional addition of browning inhibitors may increase unsafe factors. At the same time, the nature of the browning inhibitors themselves will also limit the subsequent compounding applications of kasugamycin products and increase corporate costs.

[0004] Therefore, it is a technical problem to be solved urgently to improve the stability of kasugamycin products without adding additional auxiliary agents or additives, and to keep the kasugamycin composition products stable in terms of odor, appearance or composition while ensuring safety and effectiveness by targeted control of the content of each substance in the kasugamycin composition products. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention aims to provide a stable kasugamycin product or composition. The applicant was surprised to find that the protein and polysaccharide in the kasugamycin composition have different effects on the stability of the product. By controlling the content and / or proportion of the protein and / or polysaccharide in the kasugamycin composition, the composition can be kept stable in different aspects such as appearance, odor or composition, such as no obvious color change, no significant odor or no obvious degradation of the active substance.

[0006] An aspect of the present invention is to provide a stable kasugamycin composition, based on reducing the generation of odor and / or controlling the generation of browning, the mass ratio of kasugamycin and / or its salt to protein in the composition is greater than or equal to 9:1; further, the mass ratio of kasugamycin and / or its salt to protein in the composition is greater than or equal to 10:1, or greater than or equal to 11:1, or greater than or equal to 15:1, or greater than or equal to 16:1, or greater than or equal to 17:1, or greater than or equal to 20:1, or greater than or equal to 25:1, or greater than or equal to 26:1, wherein the mass of kasugamycin and / or its salt is calculated as kasugamycin free base.

[0007] Furthermore, when the composition is a liquid, the protein content is less than or equal to 2100 mg / L, preferably less than or equal to 1800 mg / L, more preferably less than or equal to 1300 mg / L or 1000 mg / L or 800 mg / L; further, the protein content is greater than or equal to 450 mg / L, and the liquid is a kasugamycin aqueous solution or other liquid preparations.

[0008] Or when the composition is solid, the protein content in the composition is less than or equal to 3.5wt%, preferably less than or equal to 3wt%, less than or equal to 2.5wt% or less than or equal to 2wt%, based on the mass of the solid composition. Taking into account the production cost, the content of protein in the kasugamycin composition is greater than or equal to 0.65wt%, or greater than or equal to 0.7wt%, or greater than or equal to 0.75wt%, preferably greater than or equal to 0.8wt%, or greater than or equal to 0.85wt%, or greater than or equal to 0.9wt%, and the solid composition is a kasugamycin crystal product.

[0009] Furthermore, in order to reduce the hygroscopicity problem of the composition, the content of polysaccharides in the solid composition is less than or equal to 4wt%, preferably less than or equal to 3.5wt%, preferably less than or equal to 3wt%, preferably less than or equal to 2.5wt% or less than or equal to 2wt% or less than or equal to 1.5wt%, based on the mass of the solid composition. Further considering the production cost, the content of polysaccharides in the kasugamycin composition is greater than or equal to 0.4wt%, or greater than or equal to 0.45wt%, or greater than or equal to 0.5wt%, and the solid composition is a kasugamycin spray-dried product.

[0010] Another aspect of the present invention is to provide a pesticide product containing the above composition, wherein the pesticide product includes a pesticide technical, a pesticide concentrate or a pesticide preparation.

[0011] Another aspect of the present invention is the use of the above composition as a raw material in the production, processing or manufacture of pesticide products, wherein the pesticide products include pesticide technical, pesticide concentrate or pesticide formulation.

[0012] Furthermore, based on the total mass of the pesticide technical or pesticide master batch, the content of the composition in the pesticide technical or master batch is greater than or equal to 65wt%, preferably greater than or equal to 70wt%.

[0013] Another aspect of the present invention is to provide a method for preparing a stable kasugamycin composition, by reducing the content of protein and / or polysaccharide in the kasugamycin composition to stabilize the composition, wherein the mass ratio of kasugamycin and / or its salt to protein in the composition is greater than or equal to 9:1, or greater than or equal to 10:1, or greater than or equal to 11:1, or greater than or equal to 15:1, or greater than or equal to 16:1, or greater than or equal to 17:1, or greater than or equal to 20:1, or greater than or equal to 25:1, or greater than or equal to 26:1, wherein the mass of the kasugamycin salt is calculated as kasugamycin free base.

[0014] Specifically, the method for reducing the content of protein and / or polysaccharide in the kasugamycin composition comprises the following steps:

[0015] (1) Acidifying the fermentation broth with oxalic acid;

[0016] (2) Adsorb with strong acid resin and wash with pure water in reverse direction, the amount of pure water used is greater than or equal to 2 times the volume of resin and less than 6 times the volume of resin;

[0017] (3) Decolorization by activated carbon adsorption.

[0018] Preferably, the acidification pH value is about 3; further, the acidification temperature is 68°C-71°C, such as 69°C or 70°C.

[0019] Preferably, the amount of pure water for washing is greater than or equal to 2 times the volume of the resin, preferably, the amount of pure water for washing is greater than or equal to 3 times the volume of the resin, more preferably, the amount of pure water for washing is greater than or equal to 4 times the volume of the resin, and more preferably, the amount of pure water for washing is less than or equal to 6 times the volume of the resin.

[0020] Preferably, the method further comprises an organic solvent purification step, wherein the organic solvent comprises one or more of methanol, ethanol, acetone and propanol.

[0021] Preferably, the method further comprises preparing a kasugamycin solid product by cooling crystallization or evaporative crystallization to reduce the content of protein and / or polysaccharide in the kasugamycin composition, wherein the crystallization precipitation temperature is greater than or equal to 20°C; further, the crystallization cooling rate is less than or equal to 0.5°C / 10min.

[0022] Without limitation to the examples, those skilled in the art will appreciate that the purification method may further include steps such as filtration, solvent recovery, and desalting according to actual purification needs.

[0023] Furthermore, the means for reducing the content of protein and / or polysaccharide in the kasugamycin composition include one or more of the following: 1) controlling the mass ratio of kasugamycin and / or its salt to protein in the composition to be greater than or equal to 9:1, or greater than or equal to 10:1, or greater than or equal to 11:1, or greater than or equal to 15:1, or greater than or equal to 16:1, or greater than or equal to 17:1, or greater than or equal to 20:1, or greater than or equal to 25:1, or greater than or equal to 26:1; wherein the mass of the kasugamycin salt is calculated as kasugamycin free base; 2) when the composition is a liquid, the protein content is less than or equal to 2100 mg / L, preferably less than or equal to 1800 mg / L, more preferably less than or equal to 1300 mg / L or 1000 mg / L or 800 mg / L; further, the protein content is greater than or equal to 450 mg / L, and the liquid is a kasugamycin aqueous solution or other liquid preparations. When the composition is solid, based on the mass of the composition, the protein content in the composition is controlled to be less than or equal to 3.5wt%, preferably less than or equal to 3wt%, less than or equal to 2.5wt% or less than or equal to 2wt%; based on the mass of the composition, the protein content in the kasugamycin composition is controlled to be greater than or equal to 0.65wt%, or greater than or equal to 0.7wt%, or greater than or equal to 0.75wt%, preferably greater than or equal to 0.8wt%, or greater than or equal to 0.85wt%, or greater than or equal to 0.9wt%; 4) based on the total mass of the solid composition, the polysaccharide content in the solid composition is controlled to be less than or equal to 4wt%, preferably less than or equal to 3.5wt%, preferably less than or equal to 3wt%, preferably less than or equal to 2.5wt%, or less than or equal to 2wt% or less than or equal to 1.5wt%; 5) based on the total mass of the composition, the polysaccharide content in the composition is greater than or equal to 0.4wt%, greater than or equal to 0.45wt%, preferably greater than or equal to 0.5wt%.

[0024] The applicant believes that based on the technical solution and verification process described in the present invention, it is determined that proteins and polysaccharides have different effects on product stability. By purposefully controlling the content of protein and / or polysaccharides in the kasugamycin composition and the ratio relationship with kasugamycin, the kasugamycin composition has better stability in different aspects such as odor, color appearance and / or hygroscopicity, and has significant beneficial effects in storage, freight and terminal applications, and guided by the key substances that affect stability, the production process is purposefully controlled at the manufacturing end, which is conducive to cost control and improving product quality.

[0025] For the purpose of the present invention, unless otherwise specified, the terms used in this application have the following meanings:

[0026] The term "technical product" refers to a product obtained in the production process consisting of active ingredients and related impurities, to which a small amount of additives may be added if necessary. For example, kasugamycin technical product refers to a product obtained in the production process consisting of kasugamycin and / or kasugamycin salts and related impurities, to which a small amount of additives may be added if necessary.

[0027] The term "mother drug" refers to the product obtained in the production process consisting of active ingredients and related impurities, which may contain a small amount of necessary additives and appropriate diluents. For example, kasugamycin mother drug refers to the product obtained in the production process consisting of kasugamycin and / or kasugamycin salts and related impurities, which may contain a small amount of necessary additives and appropriate diluents.

[0028] The term "formulation" refers to a stable product made from pesticide technical or pesticide concentrate and suitable adjuvants, or made by biological fermentation, plant extraction and other methods.

[0029] The term "adjuvant" refers to any single component or multiple components added to pesticide products, in addition to the active ingredient, which does not have pesticide activity and active ingredient function itself, but can or helps to enhance or improve the physical and chemical properties of the pesticide product.

[0030] The term "Kasugamycin composition" refers to a mixture of kasugamycin and / or its salts with adjuvants, soluble salts, proteins and / or sugars and other substances. Based on the total mass of the "Kasugamycin composition", the content of kasugamycin and / or its salts in the "Kasugamycin composition" is 0.5%-99.5%.

[0031] The term "polysaccharide" refers to the sum of reducing sugars (glucose, fructose, lactose, etc.) and oligosaccharides (sucrose, etc.) that can be hydrolyzed into reducing sugars under the assay conditions.

[0032] The term "protein" refers to the sum of all protein contents in the raw materials for producing kasugamycin pesticide products or kasugamycin pesticide products, which can be determined using methods known in the art.

[0033] The terms “around”, “about” and “substantially” refer to numerical variations within the normal experimental or measurement error range, such as “substantially” represents an error of less than or equal to 15%, preferably an error of less than or equal to 10%.

[0034] The term "salt of kasugamycin" refers to a compound formed by combining kasugamycin with an acid ion, such as kasugamycin hydrochloride, kasugamycin sulfate, kasugamycin carboxylate, kasugamycin phosphate, kasugamycin nitrate or kasugamycin carbonate.

[0035] The term "BV" refers to the volume of the resin loaded into the resin column. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Comparative diagram of hygroscopicity of various kasugamycin solid compositions;

[0037] Figure 2 Detection liquid phase spectrum of solid composition #7;

[0038] Figure 3 Browning comparison chart of kasugamycin crystal products; Figure a and Figure b are photos of crystal product #1 before and after the accelerated experiment, respectively. DETAILED DESCRIPTION

[0039] 1. Inspection and testing methods

[0040] The reagents and solvents used in the following examples can be purchased from commercial sources or prepared by conventional methods known to those skilled in the art. All commercially available reagents and solvents were not further purified.

[0041] Protein determination:

[0042] The protein in the sample was determined by microplate reader method according to the method provided by BCA protein concentration determination kit (PC0020, Solarbio®), as follows:

[0043] 1. Prepare the working solution: According to the number of standards and samples, add 1 volume of Cu reagent to 50 volumes of BCA reagent (50:1) to prepare the BCA working solution. Mix thoroughly. The BCA working solution is stable at room temperature for 24 hours.

[0044] 2. Dilute the standard: dilute 10 μL of BSA standard with PBS to 100 μL, so that the final concentration is 0.5 mg / mL. Add 0, 2, 4, 6, 8, 12, 16, 20 μL of the standard to the protein standard wells of the 96-well plate and add PBS to make up to 20 μL.

[0045] 3. Dilute the sample 2-fold, 4-fold and 8-fold (solid samples need to be dissolved before dilution), and add 20 μL to the sample wells of a 96-well plate.

[0046] 4. Add 200 μL of BCA working solution to each well and place at 37°C for 15-30 minutes. Measure A562nm with an ELISA reader and calculate the protein concentration based on the standard curve.

[0047] Polysaccharide determination:

[0048] The anthrone colorimetric method was used to determine the polysaccharide content. The steps are as follows:

[0049] 1. Anthrone sulfuric acid solution: Take 98% sulfuric acid and distilled water to prepare 72% sulfuric acid solution. Accurately weigh an appropriate amount of anthrone (provided by Shanghai Yuanye Biotechnology Co., Ltd.), and use 72% sulfuric acid solution to prepare anthrone-sulfuric acid solution containing 0.1% anthrone.

[0050] 2. Preparation of standard curve

[0051] (1) Preparation of standard glucose solution (100 μg / ml): Dissolve 100 mg of glucose in distilled water and dilute to 1000 ml for later use;

[0052] (2) Add samples according to the specifications shown in Table 1:

[0053] Table 1 Polysaccharide standard curve sample addition specifications

[0054]

[0055] (3) Mix the added sample, place in a boiling water bath for 10 min, cool to room temperature, and compare the color at 620 nm.

[0056] (4) Use the first tube as the blank and perform colorimetry, with the optical density (OD value) as the ordinate and the sugar content (ug) as the abscissa to obtain a standard curve.

[0057] 3. Sample Preparation

[0058] Weigh an appropriate amount of solid sample or measure an appropriate amount of liquid sample. If it is a solid sample, add a small amount of water to dissolve it first; finally make the volume of the dissolved liquid or liquid sample to 50 mL, until the solution concentration is about 500μg / mL.

[0059] 4. Determination of polysaccharide content in samples

[0060] Take 0.1, 0.5 and 1.0 mL of sample solution respectively, and make up to 1.00 mL with distilled water. Add anthrone-sulfuric acid solution reagent, perform the same operation as the standard curve preparation, perform colorimetric determination, and calculate the content based on the standard curve and sample concentration.

[0061] Kasugamycin assay:

[0062] 1. Prepare 0.05 mg / mL solution of kasugamycin hydrochloride standard (provided by Xi'an Maisidi Bioengineering Co., Ltd.), detect the standard solution by high performance liquid chromatography, and calculate the kasugamycin peak area;

[0063] 2. Prepare the sample to be tested into a solution, and measure the sample solution under the same liquid chromatography conditions to calculate the kasugamycin peak area;

[0064] 3. Compare the kasugamycin peak area of ​​the sample to be tested with the kasugamycin peak area in the standard solution, and calculate the kasugamycin content in the sample to be tested.

[0065] The HPLC conditions were as follows:

[0066] The high performance liquid chromatography model was ACQUITY ARC-2489 (Waters, US);

[0067] Mobile phase: acetonitrile: sodium dodecyl sulfate solution = 1:4, v / v (pH adjusted to 2.5 with phosphoric acid). Preparation of sodium dodecyl sulfate solution: 0.8 g sodium dodecyl sulfate solid was dissolved in 400 mL ultrapure water;

[0068] Column: SymmetryShield TM RP18 3.9 x150 mm, 5 µm;

[0069] Detection wavelength: 210 nm;

[0070] Flow rate: 1.0 mL / min;

[0071] Column temperature: 35°C;

[0072] Injection volume: 10 μL.

[0073] Odor test:

[0074] Place an open beaker containing the liquid sample (100 mL) in an environment with a humidity of about 80% RH and a temperature of about 45°C, away from light and stored for 15 days. After 15 days, select 10 testers (half male and half female, aged 28-40) to score and evaluate the samples based on the odor (0-10 points). The stronger the odor, the higher the score.

[0075] The testers first sniffed odorant solutions with concentrations of 0.01wt% and 0.001wt% (provided by Shandong Huanju Technology Co., Ltd.) through the "direct sniffing method". The 0.01wt% concentration was scored as 6 and the 0.001wt% concentration was scored as 4. Then the samples were sniffed in the same way in turn. After each sniffing test, they rested outdoors for 5 minutes before the next test. Sample odor scores greater than or equal to 6 indicate that they are unacceptable for terminal application (represented as "●"), scores between 4-6 indicate that they are acceptable for terminal application (represented as "◎"), and scores less than or equal to 4 indicate that there is no impact on terminal application (represented as "○").

[0076] Browning detection:

[0077] The degree of browning of the sample is determined by measuring the change in absorbance before and after storage, and the degree of browning is displayed by the absorbance increase rate ΔA. The operation method is as follows: take 0.5g of the sample, dissolve it in 5ml of pure water, measure the initial absorbance of the solution (recorded as A0), and take the average value after measuring each sample three times. Take about 8g of the sample, grind it, sieve it through 150 mesh, spread the powder on the culture dish (thickness less than 2mm), cover the surface with plastic wrap and seal it, place the culture dish containing the powder in an accelerated test environment (50℃, continuous irradiation with fluorescent light) for 15 days, take 0.5g of each sample and dissolve it in 5ml of pure water, and measure the final absorbance of the sample after the accelerated test (recorded as A), and take the average value after measuring each sample three times.

[0078] The calculation formula of absorbance increase rate ΔA is as follows: ΔA= (A-A0) / A0; the absorbance was measured using a DR1900-02 spectrometer (Hach Lange GmbH) with an optical path of 1 cm and a wavelength of 430 nm.

[0079] 2. Implementation and Effect Experiment

[0080] Preparation of kasugamycin fermentation broth: Using Streptomyces aureus as the producing bacteria, kasugamycin fermentation broth was obtained through multi-stage fermentation in a culture medium containing low-temperature soybean cake powder, soybean oil, yeast powder, liquid sugar and other raw materials. The fermentation broth was used as the raw material for subsequent purification experiments.

[0081] Comparative Example 1

[0082] The fermentation broth was purified with reference to Example 1 of CN112679560A. The steps for preparing the purified stock solution are as follows:

[0083] (1) The fermentation liquid containing kasugamycin was acidified to a pH of about 3.5 using an acidifying agent, hydrochloric acid, and the acidification temperature was about 66°C; the acidified liquid was filtered using a nanofiltration ceramic membrane and then subjected to static adsorption to saturation using a cation exchange resin at a stirring speed of 50 rpm, and polar substances were reversely washed away using a salt-free water with a volume of 2 times the resin volume (BV), and kasugamycin was then eluted using 2.5 wt% ammonium chloride with a volume of 4 times the resin volume and a flow rate of 3 times the resin volume;

[0084] (2) Add pure water for membrane filtration, control the initial membrane pressure to 1 MPa, and stop concentration when the membrane pressure reaches 1.3 MPa;

[0085] (3) The concentrated liquid is decolorized using activated carbon fiber, and the decolorization temperature is controlled at 30°C. After 1 hour, the liquid is filtered through a liquid fine filter to obtain purified raw liquid #1.

[0086] Comparative Example 1 is an existing purification process. End users reported that the aqueous solution and technical drug prepared from the purified stock solution produced by this process had serious odor and browning after being stored for a certain period of time.

[0087] Comparative Example 2

[0088] The existing purification process was optimized: the purification method described in Control Example 1 was used to prepare the purified stock solution #2, except that the acidifier was oxalic acid, the acidification pH value was about 3, and the acidification temperature was about 70°C. The contents of protein, polysaccharide and kasugamycin in the purified stock solution were determined according to the "Testing Methods", and the mass ratio of kasugamycin to protein was shown in Table 2.

[0089] Table 2 Content of components in purified stock solutions #1 and #2

[0090]

[0091] Calculated as Kasugamycin free base

[0092] Preparation of the composition of comparative example 1-2 and odor test

[0093] Take appropriate amounts of the purified stock solutions prepared in Control Example 1 and Control Example 2, respectively, and add pure water to the purified stock solutions to prepare 2wt% (calculated as kasugamycin free base) kasugamycin aqueous solutions, recorded as aqueous solution #1 and aqueous solution #2. Determine the contents of protein, polysaccharide and kasugamycin in the aqueous solution according to the "Inspection and Detection Method". The contents of protein, polysaccharide and kasugamycin in aqueous solution #1 and aqueous solution #2, as well as the mass ratio of kasugamycin to protein are shown in Table 3.

[0094] Table 3 Content of each component in aqueous solution #1 and #2

[0095]

[0096] Calculated as Kasugamycin free base

[0097] Take 100 mL of each of the above-mentioned aqueous solutions respectively, and store them in a dark environment with a humidity of about 80% RH and a temperature of about 45°C for 15 days according to the method described in "Testing Methods - Odor Test". Then measure the odor of the aqueous solution and evaluate the odor: an odor score of 6 or more indicates that it is unacceptable for terminal application (denoted as "●"), a score between 4 and 6 indicates that it is acceptable for terminal application (denoted as "◎"), and a score of 4 or less indicates that it has no impact on terminal application (denoted as "○"). The kasugamycin content and odor score of aqueous solutions #1 and #2 after 15 days of accelerated testing are shown in Table 4.

[0098] Table 4 Kasugamycin content and odor score in aqueous solution #1 and aqueous solution #2

[0099]

[0100] Calculated as Kasugamycin free base

[0101] At present, the aqueous solution prepared by the purified stock solution produced based on the existing process has a serious odor problem. It can be seen from Tables 3 and 4 that the odor of the aqueous solution prepared by the purification process after replacing hydrochloric acid with oxalic acid and adjusting the acidification conditions is reduced, while the content of the active ingredient kasugamycin in the aqueous solution does not change much. Further analysis of the composition of the two aqueous solutions and the purified stock solution found that the content of protein and polysaccharides in the purified stock solution and aqueous solution prepared after optimization (replacing hydrochloric acid with oxalic acid and adjusting the acidification conditions) was reduced, indicating that the odor of the kasugamycin composition may be related to the content of protein and / or polysaccharides.

[0102] The purification method was further adjusted to remove or reduce the protein and polysaccharide substances, and the influence of the protein and polysaccharide content on the off-flavor of the kasugamycin composition was investigated.

[0103] Example 1

[0104] The rest of the purification process was consistent with that of Control Example 1, except that the amount of pure water (without salt water) for washing was increased or the number of times of washing without salt water was increased, with two washes (4 BV) and three washes (6 BV) respectively. The prepared purified stock solutions were recorded as purified stock solutions #3 and #4 respectively.

[0105] The contents of protein, polysaccharide and kasugamycin in the purified stock solution were determined according to the “Inspection and Testing Methods” to explore the relationship between protein and polysaccharide and the amount of water used for resin washing.

[0106] It can be seen from Table 5 that the protein and polysaccharide content in the kasugamycin stock solution will decrease with the increase of the amount of water used for resin washing. The specific adsorption effect of the resin and increasing the number of resin washings can effectively reduce the protein and polysaccharide content in the kasugamycin stock solution. However, at the same time, the amount of wastewater increases sharply, and the above purification process still has room for improvement.

[0107] Example 2

[0108] Based on the need to reduce production costs and wastewater volume, further optimization is carried out on the basis of meeting the terminal application requirements of the product:

[0109] (1) The fermentation liquid containing kasugamycin was acidified to a pH of about 3 using an acidifying agent, oxalic acid, and the acidified liquid was filtered using a nanofiltration ceramic membrane. The filtrate was then adsorbed using a cation exchange resin. At a stirring speed of 50 rpm, static adsorption was performed until saturation. The polar substances were washed away with 2 BV of salt water, and then kasugamycin was eluted with 2.5 wt% ammonium chloride, which was 4 times the amount of the resin, at a flow rate of 3 times the amount of the resin, and the eluate was collected;

[0110] (2) Purifying the eluent of step (1) with an organic solvent of ethanol: mixing ethanol and the eluent at a volume ratio of 1:1 under stirring at a speed of 200 rpm and a stirring temperature of 25° C., stirring for 1 h, and then performing plate and frame filtration to collect the solid phase; and performing a solvent recovery process on the liquid phase;

[0111] (3) dispersing the solid phase described in step (2) in 15 times the mass of deionized water, filtering by plate and frame, and collecting the filtrate;

[0112] (4) Concentrating the filtrate in step (3): adding pure water for membrane filtration, controlling the initial membrane pressure to 1 MPa, and stopping the concentration when the membrane pressure reaches 1.3 MPa;

[0113] (5) Decolorization of activated carbon fiber: control the decolorization temperature at 30°C. After 1 hour, the feed liquid is filtered through a liquid fine filter to obtain purified stock solution #5.

[0114] Purified stock solutions #6 and #7 were prepared by referring to the above method, except that the amount of salt-free water used for resin washing was 3 BV and 4 BV respectively. The prepared purified stock solutions were recorded as #6 and #7 respectively.

[0115] The contents of protein, polysaccharide and kasugamycin in the purified stock solutions #3-#7 were determined according to the “Test Methods”, as shown in Table 5.

[0116] Table 5 Content of each component in purified stock solution #3-#7

[0117]

[0118] Calculated as Kasugamycin free base

[0119] According to Table 5, it can be found that by increasing the amount of resin washing water or the number of washing times, the content of protein and polysaccharide in the purified stock solution can be effectively reduced, but the water consumption increases sharply, and the amount of wastewater and the difficulty of wastewater treatment also increase, and the production cost and sewage treatment cost are further increased. After further combining with the organic solvent purification method, the protein and polysaccharide content in the purified stock solution can be reduced and the amount of washing water can be reduced. It shows that the combination of organic solvent purification method and resin adsorption can further reduce the content of protein and polysaccharide in the purified stock solution without significantly increasing the amount of wastewater.

[0120] In addition, it was observed that, referring to the purification method described in Example 2, when the organic solvent was replaced with acetone, methanol and propanol, the purified stock solution prepared under the same conditions had substantially the same composition as the purified stock solution prepared when the organic solvent was ethanol.

[0121] Preparation of the composition of Examples 1-2 and odor test

[0122] The above #3-#7 purified kasugamycin stock solutions were used as raw materials to prepare commonly used 2 wt% kasugamycin aqueous compositions as the objects of investigation, so as to determine the odor generation of the kasugamycin aqueous compositions under different compositions and ratios.

[0123] Appropriate amounts of purified stock solutions #3, #4, #5, #6 and #7 were taken respectively, and pure water was added to the purified stock solutions to prepare 2wt% kasugamycin aqueous compositions, which were recorded as aqueous solutions #3, #4, #5, #6 and #7. Another 1L of the numbered purified stock solution #3 was taken, and 1g of glucose was added to the purified stock solution to prepare 2wt% kasugamycin aqueous compositions, which were recorded as aqueous solutions #3'. The protein, polysaccharide and kasugamycin contents of aqueous solutions #3-#7 and #3' are shown in Table 6.

[0124] Table 6 Content of each component in aqueous solution #3-#3'

[0125]

[0126] Calculated as Kasugamycin free base

[0127] 100 mL of each of the above aqueous solutions was taken, and the odor of the aqueous solutions was measured after being stored in an environment with a humidity of about 80% RH and a temperature of about 45°C for 15 days in the dark according to the method described in the "Testing Methods - Odor Test", and the odor of the aqueous solutions was evaluated according to the method described in the "Odor Test". The kasugamycin content and odor score of aqueous solutions #3-#7 and #3' after 15 days of accelerated testing are shown in Table 7.

[0128] Table 7 Kasugamycin content and odor score in aqueous solution #3-#3'

[0129]

[0130] Calculated as Kasugamycin free base

[0131] It can be seen from the data in Tables 6 and 7 that the kasugamycin content in each aqueous composition does not change much before and after 15 days. Although the polysaccharide content in aqueous solution #3' is increased by 2 times relative to aqueous solution #3, the effect on odor is small, which confirms that protein substances are the main substances for the odor generation of the kasugamycin composition, and the degree of odor is positively correlated with the protein content in the aqueous solution. At the same time, increasing the mass ratio of kasugamycin to protein can also effectively control the generation of odor, which may be related to the antibacterial property of kasugamycin itself and the threshold value of protein content required for microorganisms to produce odor. Based on actual effects and cost considerations, in the kasugamycin composition, the mass ratio of kasugamycin to protein should be greater than or equal to 9:1, preferably greater than or equal to 10:1, preferably greater than or equal to 11:1; further, the protein content is less than about 2100 mg / L, preferably less than 1800 mg / L; more preferably less than or equal to 1200 mg / L, which can effectively reduce the generation of odor. However, over-purification does not allow the kasugamycin composition to continue to show significant beneficial effects in odor control. Therefore, considering the purification cost, the protein content in the kasugamycin composition is greater than or equal to 450 mg / L, preferably greater than or equal to 500 mg / L.

[0132] Example 3

[0133] The kasugamycin product prepared by spray drying is prone to hygroscopicity, so the kasugamycin product prepared by spray drying was further investigated to determine the factors affecting the hygroscopicity of the kasugamycin product.

[0134] Take 20L of each of the purified stock solutions #1-#7 prepared in Control Examples 1-2 and Examples 1-2, and use a small spray dryer to prepare 7 types of kasugamycin solid compositions, recorded as solid compositions #1-#7. Take 2 portions of the solid composition numbered #3 (100g each), and add 500mg of water-soluble collagen (provided by Baoruyi Biotechnology Co., Ltd.) and 500mg of soluble soy protein isolate (provided by Anhui Zhonghong Bioengineering Co., Ltd.) to one portion of 100g of the #3 solid composition, recorded as solid composition #3'; add 500mg of glucose to another portion of 100g of the #3 solid composition, recorded as solid composition #3''. The contents of kasugamycin, polysaccharides and proteins in the above 9 solid compositions were determined according to the method described in the "Inspection and Detection Methods", as shown in Table 8, among which the detection liquid phase spectrum of solid composition #7 is shown in Table 8. Figure 2 shown.

[0135] Table 8 Content of each component in the solid composition

[0136]

[0137] Calculated as Kasugamycin free base

[0138] Hygroscopicity test of Example 3

[0139] 50 g of each of the nine kasugamycin solid compositions prepared in Example 3 were taken and placed in an environment with a humidity of about 80% RH and a temperature of about 25° C. The quality of the solid compositions was tested on the 1st, 3rd, 5th, 7th, 12th and 15th day, and the kasugamycin content on the 15th day was tested. Figure 1 and as shown in Table 9.

[0140] In actual preparation production, since the spray product needs to undergo processes such as crushing and mixing, the hygroscopicity of the original drug not exceeding 10% has no significant effect on the preparation quality control, and the hygroscopicity not exceeding 5% has no significant effect on long-term storage and transportation (more than 1 month).

[0141] according to Figure 1 As can be seen from Table 9, through the hygroscopicity test of different kasugamycin solid compositions, the applicant found that the effect of protein on the hygroscopicity of kasugamycin solid compositions is limited, while the content of polysaccharides will significantly affect the hygroscopic properties of the solid composition. In particular, after the solid composition #3'' specifically increased the polysaccharide content, the hygroscopic weight gain reached 10% on the 12th day, reaching 55 g, and 56.2g on the 15th day. The hygroscopic weight gain of the solid composition #3' with specific increase in protein content is not obvious, which may be related to the large amount of alcohol hydroxyl hydrophilic groups contained in the polysaccharide. Similarly, the solid compositions #1 and #2 with higher polysaccharide content all exceeded 10% on the 12th day, and the kasugamycin content of the active ingredient in the solid composition was also reduced to about 65%. The hygroscopic mass change of the solid composition is shown in Table 9.

[0142] Table 9 Changes in moisture absorption mass of solid compositions

[0143]

[0144] Calculated as Kasugamycin free base

[0145] In summary, based on the total mass of the solid composition, the polysaccharide content in the solid composition is controlled within about 4wt%, and the moisture absorption and weight gain of the kasugamycin composition of solid composition #3 on the 12th day is not more than 10%, which effectively reduces the risk of product instability caused by rapid moisture absorption of the original drug raw material when it is required for short-term storage such as internal transportation in the factory and preparation processing. The polysaccharide content in the solid composition is further controlled within about 1.5wt%, that is, the weight gain of solid composition #5 does not exceed 5% even after 15 days, which meets the relevant quality control requirements of the enterprise and meets the medium- and long-term storage and shelf life requirements. However, the hygroscopicity of the solid composition is not significantly reduced by further reducing the polysaccharide to about 0.4wt%. Based on hygroscopicity control and cost considerations, it is not easy to over-purify to reduce the polysaccharide content in the solid composition, such as the polysaccharide content should be controlled to be greater than or equal to 0.4wt%, preferably greater than or equal to 0.45wt%, preferably greater than or equal to 0.5wt%.

[0146] Example 4

[0147] Kasugamycin technical is mainly composed of kasugamycin crystal products. However, at present, kasugamycin technical will gradually produce browning phenomenon with the extension of storage time. Therefore, it is necessary to further use kasugamycin crystal products as the object to determine the influencing factors causing browning in kasugamycin technical or crystal products.

[0148] Take 10L of each of the purified stock solutions #1-#7 prepared in Control Example 1-2 and Example 1-2, and concentrate the purified stock solution to 5L by reduced pressure evaporation at 50°C, and maintain the temperature. After that, the purified stock solution was crystallized, cooled and crystallized at a cooling rate of 0.5°C / 10min, and the crystallization temperature dropped to 20°C. After crystallization for 12 hours, each crystal product was collected and dried at room temperature to obtain kasugamycin crystal products, which were recorded as crystal products #1-#7. The protein, polysaccharide and kasugamycin contents in each crystal product were determined by the method described in the "Testing Methods", as shown in Table 10.

[0149] Table 10 Content of each component in the crystallized product

[0150]

[0151] Calculated as Kasugamycin free base

[0152] Browning test of Example 4

[0153] According to the method described in "Inspection and Detection Method - Browning Detection", the 7 crystalline products prepared in Example 4 were placed in an accelerated environment (50°C, continuous irradiation with fluorescent lamps for 15 days) for detection, and the absorbance of the samples before and after the 15-day experiment was measured. The absorbance on day 0 was A0, and the absorbance after the 15-day accelerated experiment was A. The absorbance change rate ΔA was calculated. The change in absorbance of the crystalline product before and after the 15-day accelerated test and the kasugamycin content on the 15th day are shown in Table 11.

[0154] From the perspective of actual production and processing needs, when the color change (calculated by absorbance) of the original drug is less than or equal to 15%, it can be considered that the original drug has not undergone obvious browning and meets the relevant enterprise standards.

[0155] Table 11 Absorbance changes of crystallized products

[0156]

[0157] Calculated as Kasugamycin free base

[0158] According to Table 11, the content of effective kasugamycin in the crystallized product did not change much before and after the 15th day of the accelerated experiment. The initial state of the above 7 crystallized products was white powder, and there was no significant difference in color between the powders; after 15 days of the accelerated experiment, the color of the crystallized product #1 changed significantly and finally became light yellow brown (such as Figure 3 As shown), crystallized product #2 is light gray-brown, and other crystallized products do not change color significantly. In addition, it can be seen from Table 11 that the absorbance change rate of crystallized products #1 and #2 exceeds 15% of the enterprise standard. Due to the high protein content in crystallized products #1 and #2, and the polysaccharide content in #1 and #2 is not much different from #3- #7, the obvious browning of crystallized products #1 and #2 should be related to the higher protein content in the crystallized products. Controlling the protein content in the kasugamycin composition within 3.5wt% can prevent the significant color change of the kasugamycin composition caused by the browning reaction, and when the protein content is reduced to 0.65wt%, the browning phenomenon has not been observed. Comprehensive product actual effect and cost and other factors, the content of protein in the kasugamycin composition should not be greater than or equal to 3.5wt%, and further based on the production cost control needs, the protein content can be greater than or equal to 0.65wt%.

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0160] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

Claims

1. A method for preparing a stable kasugamycin composition, characterized in that: The following steps are involved: (1) Acidifying the fermentation broth with oxalic acid; (2) Adsorb with strong acid cation exchange resin and wash back with pure water, the amount of pure water used is greater than or equal to 2 times the volume of the resin and less than or equal to 6 times the volume of the resin; (3) Decolorization by activated carbon adsorption; The method further comprises the step of purifying the fermentation broth with an organic solvent, specifically purifying the fermentation broth after being treated in step (1) and step (2), wherein the organic solvent comprises one or more of methanol, ethanol, acetone and propanol; The mass ratio of kasugamycin and / or its salt to protein in the composition is greater than or equal to 9:1, wherein the mass of the kasugamycin salt is calculated based on kasugamycin free base; The composition is liquid, has a protein content of less than or equal to 2100 mg / L, and a protein content of greater than or equal to 450 mg / L; or, The composition is solid, and has a protein content of less than or equal to 3.5 wt % and a protein content of greater than or equal to 0.65 wt % based on the mass of the composition; or The composition is solid, and based on the mass of the composition, the polysaccharide content in the composition is less than or equal to 4wt%, and the polysaccharide content in the composition is greater than or equal to 0.4wt%.

2. The preparation method according to claim 1, characterized in that: The method may further comprise the steps of plate and frame filtration and / or concentration.

3. The preparation method according to claim 1 or 2, characterized in that: The fermentation broth of step (1) is acidified to a pH of 3.

4. The preparation method according to claim 1 or 2, characterized in that: The organic solvent is mixed with the fermentation liquid treated in step (1) and step (2) at a volume ratio of 1:1.

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